Macrocyclic bifunctional chelators will be synthesized and conjugated to engineered antibodies and antibody fragments for tumor imaging and therapy. DOTA will be used as a model macrocycle that binds a variety of radiometals, regardless of their intrinsic coordination chemistry requirements. Radiometals for imaging include 111In for conventional imaging using whole antibodies, and 64Cu for PET using antibody fragments, while 90Y will be used for radioimmunotherapy. The use of novel linkers, site specific conjugation, and different chemical linkers will be used to minimize normal tissue uptake, especially the kidney and liver, while retaining high rates of radiometal loading and high tumor uptake. Specific Aim 1 is to optimize peptide linkers that have C-terminal basic residues that allow peptide bond cleavage between the chelate and antibody linkages by brush border carboxypeptidases in the kidney tubules. Preliminary data suggests that this approach can lower kidney uptake by as much as 50 percent. In Specific Aim 2 we plan to explore the effects of site specific conjugation methods on the metabolic clearance of both radiometal (especially 64Cu) and 18F labeled antibody fragments. This study is exemplified by the macrocycle DO3A which is coupled with vinyl sulfone derivatives and then conjugated to antibody fragments either through cysteine or lysine residues at pH 7 or pH 9, respectively. In Specific Aim 3 we will develop and characterize novel DOTA analogs such as the tetrahydrazide (DOTH), tetrathioamide, and tetrathiolate that are expected to load radiometal at faster rates than DOTA and offer unique routes of metabolic clearance that have the potential to decrease kidney and liver retention of radiometal. Preliminary data show that DOTH has three times the Y3+ loading rate of DOTA, and can be conjugated to antibody fragments either via vinylsulfone or hydrazone chemistries. The overall approach is expected to lead to clinical products with improved radiometal labeling and tumor to normal tissue ratios, thus improving the performance of both tumor imaging and tumor therapy.